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Alkylation of nucleic acids by DNA-targeted 4-anilinoquinolinium aniline mustards: Kinetic studies
223
Chem.-Biol. Interactions, 77 (1991) 223--241 Elsevier Scientific Publishers Ireland Ltd.
ALKYLATION OF NUCLEIC ACIDS BY DNA-TARGETED 4-ANILINOQUINOLINIUM ANILINE MUSTARDS : KINETIC STUDIES
CHARMIAN
J. O ' C O N N O R a, W I L L I A M A. D E N N Y b and JUN-YAO F A N a
aDepa~tment of Chemistry and bcct~bCerResearch L ~ , Auckland, P~i~;te Bog, Auckland (New Zealand)
School of Medicine, U~iversity of
(Received July 31st, 1990) (Revision received October 19th, 1990) (Accepted October 19th, 1990)
SUMMARY
The rate constants for hydrolysis of a series of 4-substituted aniline mustards Ar-X~pC~H4-N(CH2CH 2C1)2, where Ar is 4-anilinoquinolinium and X = O, CH2, CONH and CO, have been measured in water and 0.02 M irnidazole buffer at 37°C and in 50% aqueous acetone at 66°C. The equilibrium binding constants of the compounds and their hydrolysis products to nucleic acids of differing base composition have been determined at varying ionic strengths, and the results are consistent with the compounds binding as expected in the DNA minor groove. The alkylating reactivity of the mustards towards these nucleic acids has been measured in water at 37°C and in 0.01 M HEPES buffer over a range of temperatures from 25°C to 60°C. Evaluation of the thermodynamic parameters for these kinetic and equilibrium studies suggests that the interaction with nucleic acids is via an internal SN2 mechanism involving an aziridinium ion.
Key wo~ds: Aromatic nitrogen mustards -- Nucleic acids -- Entropy of activation -- Alkylation -- Association constants -- Binding
INTRODUCTION
Bifunctional alkylating agents are an important class of anticancer drugs [1], which express their cytotoxic and antitumor effectsby cross-linkingcellularDNA [2].The most common alkylatingmoiety employed isthe N-bis(2-chloroethyl)aniline functionality. Although the cytotoxic (and therapeutic) effects of these compounds are due to the formation of DNA cross-links (primarily between guanine N7 posiCorrespondence to:Charmian J. O'Connor, Department of Chemistry, Universityof Auckland, Private Bag, Auckland, N e w Zealand.
000~2797/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd.
224 tions [3]), the degree of cross-linking by simple mustards is low. The majority of DNA alkylation lesions are monoadducts, which are considered to be genotoxic (mutagenic) rather than cytotoxic [4]. There is interest in the concept [5,6] of targeting alkylating agents to DNA by attaching them to DNA-affinic carrier ligands, either intercalators [7--9] or minor groove binding moieties [10,11]. The aims of such an approach include increasing drug potency [7,12], avoiding some of the common mechanisms of cellular resistance to alkylating agents [13], and altering the pattern of DNA lesions formed [14,15] and their repair. Recent results demonstrate the usefulness of this approach, which has been shown to increase drastically the in vitro cytotoxicity of simple aniline mustards [7,11] and also significantly alter the regioselectivity of their alkylation of DNA [11,16]. In particular, targeting with minor groove binding ligands has been shown to direct away from the guanine N7 site preferred by simple alkylators to the adenine N3 site in the minor groove [11]. We have recently reported the synthesis and biological evaluation [17], and the kinetics of hydrolysis and alkylation of 4-(4'-nitrobenzyl)pyridine [18], of a series of aniline mustards (1) based on the DNA minor groove binding [19] 4-anilinoquinoline derivative (2). However, these compounds were not sufficiently water-soluble for DNA studies. In order to ascertain in more detail the mode of interaction of compounds of this general class with DNA, we prepared the more soluble quinolinium quaternary salts (3--6), and now report studies of the interaction of these compounds with nucleic acids. We have studied the kinetics of their alkylation of a variety of native nucleic acids, and have measured their equilibrium binding constants over a range of temperatures and ionic strengths. A minimum of two parameters, the intrinsic association constant, K, and the density of binding sites in the polynucleotide, n ' , is required to describe the interaction. The values of these parameters are sometimes dependent upon the model chosen to describe the interaction. In this investigation we have used both the Scatchard [20] and the McGhee and von Hippel [21] models and find the results therefrom very comparable. MATERIALS AND METHODS
Materials Double-stranded calf thymus DNA, yeast type VI RNA and synthetic poly(dGdC) and poly(dAdT) were purchased from Sigma. Micrococcus lysodeikticus DNA was purchased from Miles Laboratory Inc. Denatured DNA was prepared immediately before use by boiling the native DNA for 5 min and then cooling rapidly on ice. The concentration of nucleic acid was determined spectrophotometrically. Molar absorptivities at 260 nm are reported as 6600 M-1 cm-1 for calf thymus and M. lysodeikticus DNA [22], 8160 M -1 cm -~ for RNA [23], and 7100 M -~ cm -~ and 6600 M -1 cm -~ for poly(dGdC) and poly(dAdT), respectively [24]. Imidazole was from BDH and N-2-hydroxyethylpiperazin-N'-2-hydroxyethylpiperazin-N'ethanesulfonic acid, HEPES, was from Serva. The pH of HEPES buffer solutions was adjusted with HC104.
225
l~I/ CH2CH2Cl
~ I
HCl
1: X=O,CH2,CON~I,CO
t
CH3 2
~
~
~/~
/ CH2CH2cl
~
CH2CH2Cl
I
M~F3SO3
x 3
O
4
S
CH2 CONH
6
co
The trifluoromethanesulfonate salts of the quinolinium mustards (3--6) were prepared as described elsewhere [17]. Stock solutions were made up in methanol, and were stored at - 15°C. The initial (bifunctional) N,N-his(2-chloroethyl) compounds are referred to below as 'mustards', the half-hydrolysed (monofunctional) N(2-chloroethyl)-N(2-hydroxyethyl) compounds as 'monomustards' and the N ~ rbis(2-hydroxyethyl) compounds as 'diols'.
Apparatus Absorption spectra were measured on a Cary 219 spectrophotometer and fluorescence spectra on a Shimadzu RF540 spectrofluorophotometer, both interfaced to an Apple IIe computer. ~ P L C measurements were made using a Micromeritics 7600 HPLC Gradient System with a C-18 10.0 ~m reverse phase 250 x 5.0 mm column, and were analyzed using a Digital Solutions Delta Junior chromatography data system.
226
Rate constants of hydrolysis of mustards A 100-~1 sample of 0.16 mM mustard in methanol was added to 1 ml distilled water held at 37°C. At various time intervals aliquots (20-~1) were withdrawn and analyzed by HPLC at k 260 nm. The eluting solvent was a 1:4 mixture of solvent A (10% CH3CN + 90% buffer (1 M NH4OAc + 10 mM heptane sulfonate + 10 mM triethylamine, pH 5.00)) and solvent B (90% MeOH + 10% CH3CN) and the flow rate was 0.5 ml min -1. The rate constants kl and k2, for hydrolysis of the mustard to monomustard and of monomustard to diol, respectively, were calculated from computation of the peak areas of the three species. Details of the analysis are presented elsewhere [18]. The hydrolytic reaction was also carried out in 0.02 M imidazole buffer at ionic strength 0.025. The pH was adjusted with HC104 and the ionic strength maintained with NaC104. Under these conditions the hydrolytic products tend to precipitate (as seen by diminished HPLC peaks and changed absorbance spectra) and thus only the value of k I could be calculated. The retention times of the mustards and their hydrolysis products are given in Table I. These show unequivocally that, in completely degraded solutions of the mustards, both in water alone and in buffer systems, there is no peak corresponding to the retention time of the mustards. In other words, there is no interference from degradation products with the assay for intact mustards.
Alkylation of nucleic acids by mustards A 100-~1 sample of 0.16 mM nitrogen mustard in methanol was added to 1 ml of an aqueous solution (milli-Q water) of nucleic acid whose concentration had been determined from its absorbance at 260 nm [22,23]. The reaction was carried out at 37°C. During the incubation period (calculated to allow for 100% hydrolysis in the absence of nucleic acid) 100-~l aliquots were withdrawn, mixed well with an equal volume of butanol and centrifuged for a few seconds to separate the two phases. A 20 ~l sample of the upper {butanol) phase was then analyzed by HPLC using the same 1:4 mixture of solvent A and solvent B described above. This separation procedure removed nucleic acid components from the injection phase. During the course of the reaction three peaks (for mustard, half-mustard and diol) were observed, but the total peak area decreased rapidly. Division of the peak area of the diol at the end of the reaction by the initial peak area of the TABLE I RETENTION TIMES OF MUSTARDS AND THEIR HYDROLYSIS PRODUCTS a Compound
Mustard (min)
Monomustard (min)
Diol (min)
3 4 5 6
17.0 12.6 13.7 15.6
12.6 10.3 11.4 12.9
11.2 9.5 9.6 10.2
aRetention time of solvent 0auffer/water/MeOH) = 7.5--8.3 min.
227 mustard gave a value for extent of hydrolysis. This calculation depends on the assumptions that both the extraction coefficients and the UV absorbances of mustard and diol are equal. Calculation of the rate constant for loss of mustard, k~, gives the sum of the rate constants for hydrolysis and alkylation of nucleic acid. Rate constants of formation of alkylated products Alkylation of certain DNA bases by nitrogen mustards produces fluorescent products [25,26], and a binding assay was devised which was based on the change in fluorescence due to the DNA adducts. Suitable emission and excitation wavelengths were chosen by comparison of the spectrum of unreacted sample with that of a sample where the mustard had been allowed to react completely with the nucleic acid (5 h at 37°C, 300 ~l of 0.16 mM mustard in MeOH added to 3 ml nucleic acid). The rate of formation of aikylated products was then monitored throughout the reaction period and the experimental data treated by RF-540 spectrofluorophotometer control software to calculate values of k~k. Spectrophotometric titrations To 3 ml of a 1-~M solution of drug (mustard or diol, the latter being formed by complete hydrolysis of the corresponding mustard in milli-Q water and monitoring by HPLC) in a cuvette were added, sequentially, - 2-~1 aliquots of a concentrated solution ( - 1.2 mM basepair) of nucleic acid containing the drug at the same concentration as that in the cuvette. The total drug concentration was thus maintained at a constant value. After addition of each aliquot the absorption spectrum (220--500 nm) was recorded on a Cary 219 spectrophotometer. Addition of the aliquots was continued until either the absorbance became constant or the increasing concentration of nucleic acid itself caused a slight increase in absorbance. These titrations were repeated over a range of temperatures for binding of mustards 3-6 and at 25°C for binding of the corresponding diols of 3-6 to doublestranded calf thymus DNA, and at 19°C for binding of 3 to a number of other nucleic acids. The maximum amount of hydrolysis that would have occurred during these titrations was 5%. The Scatchard model The Scatchard [20] equation: v/C~ = Kv + Kin'
(1)
in which v is the binding ratio, C~ the concentration of free ligand, K the intrinsic association constant for the binding of the ligand to a single site in the lattice and n' the site size expressed in nucleotide residues in terms of base pairs, v and C~, determined from spectrophotometric titrations, were calculated by use of Eqns. 2:--4. (75 ffi (Af - Ax)Ct/(Af - Ab)
(2)
228 v
= Cb/N
(3)
Cb
Cf-- C t -
(4)
where Cb and Ct are the concentrations of drug bound to nucleic acid and total drug, respectively; N is the concentration of nucleic acid; and At, A b and A Xare the absorbances of the free and bound drug and the test sample, respectively. Scatchard plots of v/Cf against v yield a gradient equal to K and an x-intercept equal to 1In' The McGhee and yon Hippel model This model is used in situations where a bound ligand covers two or more lattice residues. The simple Scatchard relationship [21] does not allow for this complication since it is valid if the ligand binds to only one repeating unit, e.g. to one base or one base-pair. The McGhee and von Hippel analysis (Eqn. 5) ~ / c f --
K(1 - n'~) n' [1 -
(n'
-
1)p] n ' - I
(5)
allows for the effect of neighbouring site exclusion on binding equilibria. When n' > 1, a plot of p/C~ against v will be curved and the values of K and 1/n' are determined from the intercepts of the curve on the y- and x-axes, respectively. Calculation of effective electric charge on mustard 3 The method of Record et al. [27,28] (Eqn. 6) uses 6InK 61n[Na+]
-Z¢
(6)
in whichK is the observedequilibriumconstant for bindingof 3 to double-stranded calf thymus DNA, [Na+] is the sodiumion concentration of NaCI or NaCIO, and ¢ is the fraction of counter ions associated with each DNA phosphate. A plot of InK against ln[Na+] has a slope of -Z¢ where Z is the effective electric charge on the drug. For doublestranded DNA, ¢ -- 0.88.The alternativemethodof Wilson and Lopp [29] (Eqn.7) 5InK -- - 2 n ' ( ¢ - ¢*) - Z*¢ 51n[Na ÷]
(7)
was also used to calculate Z*, the effective electric charge on 3. In this model, n" is the number of neighbouring sites excluded upon ligand binding and ¢* is the fraction of counter ion per phosphate in the intercalated drug-DNA complex. Average values of n' = 3.0 antiC* -- 0.82 have been used in this investigation. RESULTS AND DISCUSSION
Rate constants of hydrolysis in the absence of nucleic acid While there have been extensive studies of the hydrolysis rates of aniline
229
mustards [7,30], including the non
(8)
logk2 = -3.90% - 4.30 n = 4, r = -0.92
(9)
The substantial negative slopes of these lines are indicative of assistance by electron-donation to the reaction centre. The slopes of equations describing hydrolysis of the hydrochloride salts at 66 °C in 50% aqueous acetone were comparable (-2.56 and -2.78 for k 1 and k 2 respectively). The rate constants for the first hydrolysis step (mustard to monomustard) were independent of pH in the range 6.35--7.25 in imidazole buffer, where the pH was adjusted by addition of HCIO 4 while maintaining constant ionic strength with added NaCIO 4. Measurement of the rate constant for the second hydrolysis step (monomustard to diol) was not possible because of precipitation of the hydrolysis products in the buffer medium as the reaction proceeded.
Binding to nucleic acids Association constants for binding to nucleic acids were measured specT A B L E II R A T E CONSTANTS OF HYDROLYSIS OF M U S T A R D S 3 TO 6 AT 37°C (AND OF THEIR C O R R E S P O N D I N G H Y D R O C H L O R I D E SALTS IN 50% A Q U E O U S A C E T O N E AT 66°C |18]) Compound
Solvent
pH
104 k I (s-1)
3 3 3 3 3 4 5 6
buffera buffera buffera buffera H20 H20 H20 H20
7.25 6.95 6.65 6.35
1.91 (3.69) 1.93 (3.59) 1.84 (3.68) 1.92 (3.62) 2.71 (3.15) 2.03 (2.79) 1.48 (3.26) 0.0146 (0.0353)
80.02 M imidazolebuffer, ion strength (NaClO4) ffi 0.025.
104 k 2 (s-i)
3.40 (2.50) 1.49 (2.79) 1.94 (7.01) 0.00365 (0.0694)
230
trophotometrically. Figure la shows a set of typical spectra obtained for the titration of 3 with calf thymus DNA. Addition of nucleic acid causes a bathochromic shift and a decrease in absorbance at km~ (350 nm) of the drug (Fig. lb). The presence of a clear isosbestic point in Fig. la provides further evidence for the formation of a drug/nucleic acid complex. The binding ratio ~ (tool drug bound/mol base pairs), and the free drug concentration C~were determined for mustards 3--6 in the presence of varying concentrations of calf thymus DNA at temperatures 19°C, 25°C and 37°C and in HEPES buffer at 22 °C and for mustard 3 in the presence of a variety of nucleic acids at 19°C. The results for typical binding isotherms for 3 are shown in Fig. 2. The
a ta. m9 -d • . ee ,.] ~_;a. BT"J
l,k?
I'¢//2"%
l,¢/// u
1
~.RI!
4eQ Ni.vml Inl~lth(nm) 0.800 b
i
0.60C
Ih w
0.400
0
I
10
I
20
i v
A v
,
30
A w
,
40
T
5O
Fig. 1. (a) Spectra of 3 (1 ~M) obtained upon titration with 2-~1 aliquots of 2 mM calf thymus DNA. (b) Plot showing the effect of increasing concentration of calf thymus DNA on the absorbance of 3 at X 350 nm.
231 3
d 2
1
I
I
b ~
2
0
I
I
!
.
I
I
,
u
!
I
C
f
1
a
0.1
0.2
0.3
V
0.4
0
0.1
O
g
0.2
0.3
"
I
0.4
,
0.5
V
Fig. 2. Scatehard plots for binding of 3 to nucleic acids: (a) double-stranded calf thymus DNA; (b) single-stranded calf thymus DNA; (c) double-strandedM. lysode~lct~s DNA; (d) yeast RNA; (e) po-
iy(dADT) and (f) poly(dGdC).
solidlinesdrawn through the data represent the best fitusing the Scatchard equation (Eqn. 1) [20].Parameters used to fitthis equation to the experimental data are provided in Table III. The interpretation of such data depends on the theoretical model used. Since the simplest situation [20] is considered in these studies,and the data are assumed to pertain to simple mass-action interactionbetween the drug and discretenoninteracting sites spaced along the nucleic acid chain, straight lines can be fitted (e.g. by the method of least squares) to the data points and the interaction parameters K and n' can be calculated.Under identicalbinding conditions,3 binds more strongly to double-stranded calf thymus D N A than it does to the singlestranded nucleicacid.There is also a base sequence preference, since 3 binds most strongly to poly(dAdT), least strongly to poly(dGdC), and more tightly to calf thymus DNA (50°70AT) than to M. lysodeikticus DNA (3007oAT). This is evidence that the mustards 3--6 are indeed minor groove binding ligands like the parent molecule (2), since such compounds bind preferentially to AT-rich regions in doublestranded DNA [19]. The drug binding site size n' remains essentially constant
3
3
Single-stranded calf thymus DNA
Double-stranded
.2.50 4.23 2.39
3.11
1.68
3.36 4.54 3.46 5.18
(M- 1)
19°C 10-SK
(4.0)c 2.4 2.2
2.7
(2.8) c
3.0 3.3 2.9 3.0
n ' (n)
aValues determined using the McGhee and yon Hippel model. bValues determined in 0.01 M Hepes buffer at pH 7.0. CThe site size expressed in terms of base nucleotide residues.
Yeast RNA poly(dAdT) poly(dGdC)
DNA
3 3 3
3 4 5 6
Double-stranded calf thymus DNA
M. lysodeikticus
Drug
Nucleic acid
3.01 5.88 4.32
4.89
2.21
4.99 5.55 5.09 6.72
(M 1)
19°C a 10-6//"
(3.8)¢ 2.2 2.1
2.4
(3.1) ¢
3.3 3.4 3.2 3.3
n' 2.86 3.83 3.00 4.11
(M 1)
25°C 10-SK 2.9 2.8 2.9 2.8
n' 2.17 3.00 2.46 3.14
(iV[- 1)
37°C 10-SK
2.4 2.4 2.8 3.3
n'
0.635 1.03 0.694 1.11
(M- 1)
22°C b 10-SK
The parameters were determined at all temperatures using the Scatchard model and at 19°C using the McGhee and yon Hippel model.
2.2 2.6 2.7 2.9
VALUES OF THE INTRINSIC ASSOCIATION CONSTANT (K) AND THE SITE SIZE E X P R E S S E D IN NUCLEOTIDE RESIDUES ( n ' ) FOR BINDING OF MUSTARDS TO NUCLEIC ACIDS
TABLE III
O~ t~
233 TABLE IV THERMODYNAMIC PARAMETERS DERIVED FROM E F F E C T OF TEMPERATURE ON THE INTRINSIC ASSOCIATION CONSTANT FOR BINDING OF MUSTARDS TO DOUBLESTRANDED CALF THYMUS DNA Drug
AH ° (kJ tool -1)
AG ° (kJ mo1-1)
AS ° (J mo1-1 K -1)
3 4 5 6
-18 -17 -14 -20
-37 -38 -37 -38
62 68 77 58
at 2.6 ± 0.4 nucleotides per binding site with all the nucleic acids, suggesting no change in the mode of binding. Table III also gives values of the binding parameters of these drugs to nucleic acids at 19°C determined using the McGhee and yon Hippel equation (Eqn. 5). These values of K (although a little larger) show similar trends to those calculated from the Scatchard model and the values of n are very comparable (2.7 ± 0.6). For the compounds binding to calf thymus DNA plots of lnK (K determined using the Scatchard model) against 1/T were linear, and from these van't Hoff plots values of the enthalpy of interaction, zk/-/°, were calculated (Table IV). The values of ~-/° and the derived values of the Gibbs free energy of activation, ~G ° were negative for all the mustards 3-6. Other work has shown that both DNA intercalating agents (e.g. ethidium bromide, proflavin [32] and daunomycin [33]) and minor groove binding agents (e.g. netropsin and analogues [34l) show negative enthalpies of binding. The values for the entropy of binding of 3-6 with DNA are very large and positive (Table IV). This is in agreement with other studies of minor groove binders [34], and is consistent with the large binding constants. It is well-known that raising the ionic strength of the medium decreases the value of the binding constant of small molecules to DNA [22--24,35,36]. The values of K given in Table V show that this decrease is also true for 3 when bound to TABLE V E F F E C T OF IONIC STRENGTH ON THE INTRINSIC ASSOCIATION CONSTANT FOR BINDING OF 3 TO DOUBLE-STRANDED CALF THYMUS DNA AT 20°C [ Salt] (M)
10- 5K (M - 1)
Z
Z*
0.025 M NaC1 0.010 M NaCl 0.005 M NaCl
2.68 7.64 12.0
1.1
0.66
0.025 M NaCIO 4 0.010 M NaCIO 4 0.005 M NaCIO 4
0.641 2.48 13.6
2.1
1.7
234
calf thymus DNA. Analyzing these data by the method of Record et al. [27,28] or the method of Wilson and Lopp [29] allows calculation of the value of the effective electric charge on the drug, Z or Z*, respectively, and these are given in Table V. The two models provide consistent results, demonstrating that the effective charge on the drug when it binds to the nucleic acid is dependent upon the salt which is used to adjust the ionic strength. In the presence of NaC1, the drug carries a charge between 0.66 and 1.1, but when NaClo 4 is used the charge is between 1.7 and 2.1. The compounds are methylquinolinium salts, and thus carry one permanent charge on the quinoline nitrogen. We have previously shown [18] that chloride ions markedly inhibit the formation of the aziridinium ion, thereby preventing an increase in effective electric charge. Conversely, in the presence of perchlorate ions, the values of Z are close to 2, indicating that under these conditions formation of a partial positive charge on the aziridinium ion is promoted.
Rate constants for alkylation of nucleic acids Table VI shows the derived rate constants kr for loss of the mustards in the presence of a variety of nucleic acids, measured by HPLC. The values of k~ are equal to the sum of the rate constants for alkylation of the nucleic acid and hydrolysis of the mustard, since they are derived from the loss of area in the mustard HPLC peak as the reaction progressed. Also given are the values for the percentage hydrolysis occurring at the completion of the reaction. As with TABLE VI RATE CONSTANTS FOR LOSS OF MUSTARD BY HYDROLYSIS AND/OR ALKYLATION (kr) AND VALUES FOR THE EXTENT OF HYDROLYSIS OF MUSTARDS IN NUCLEIC ACID SOLUTIONS AT 37°C Nucleic acid
Double-stranded calf thymus DNA a Single-stranded calf thymus DNA a Double-stranded M. lysodeikticus DNA b Single-stranded M. lysode/kt/cus DNA b Yeast RNA c poly(dAdT)d poly(dGdC)d a[DNA] b[DNA] C[RNA} d[dAdT]
104k~ (s-1) hydrolysis (%) 104k~ (s-1) hydrolysis (%) 104kE (s- 1) hydrolysis (%) 104kr (s-1) hydrolysis (%) 104kE (s-1) hydrolysis (%) 104k~ (s- 1) hydrolysis (%) 104kr (s- 1) hydrolysis (%)
= 0.70 mM base. ffi 0.58 mM base. ffi 1.0 mM base. ffi [dGdC] = 0.52 mM base.
3
4
5
2.62 12.4 0.869 10.9 1.85 25.0 1.94 14.3 1.80 4.2 2.34 0 2.44 39.0
0.565 16.8 0.228 8.8 1.23 31.9 1.09 15.1 1.00 1.4
0.887 15.2 0.690 9.7 1.46 26.7 1.24 13.2 0.775 2.0
6
0.0106 9.9
0.0329 13.8
0.509 0.2
235 the values of the rate constants for hydrolysis alone (k1 and k2), the values of k r also correlate well with Hammett parameters, giving in fact almost identical equations (Eqns. 10 and 11). For hydrolysis/alkylation of double-stranded calf thymus DNA (AT/GC ratio 50:50) in water:
(10)
logkr ffi -2.94% - 4.42 n = 4, r ffi - 0 . 9 2
while for double-stranded M. lysodeikticus DNA (AT/GC ratio 30:70) in water:
(11)
logkr = - 2 . 4 1 % - 4.18 n ffi 4, r = - 0 . 9 3 and for yeast RNA in water: logk, = - 0 . 3 9 % - 4.09 n ffi 4, r = - 0 . 9 7
(12)
Since alkylated adducts of guanosine [26,37] (and to a lesser extent adenosine [25]) have fluorescent properties, the formation of these adducts may be monitored by spectrofluorophotometry, as an alternative measure of DNA alkylation alone. Although this method of analysis probably does not detect reaction at other bases, it does provide information about relative rates of reaction within the series. The values obtained by this method (Table VII) for the rates of alkylation by compounds 3 and 5 agree well with those determined above by HPLC. These data show that double-stranded calf thymus DNA is alkylated about 2--3-fold as rapidly as when single-stranded, but at about the same rate as RNA.
T A B L E VII R A T E C O N S T A N T S F O R F O R M A T I O N (kALK) OF F L U O R E S C E N T DNA-ADDUCTS AT 37QC Nucleic acid
3
5
Double-stranded calf thymus DNA
)~ex (nm) )~em(nm) 104kalk (s-1)
309 410 2.09
310 405 0.654
Single-strandedcalfthymus D N A
)~ex (nm) )~em(nm) 104kalk (s- 1)
310 410 1.19
310 420 0.201
Yeast RNA
kex (nm) kern (rim) 104kalk (s- 1)
308 412 2.23
308 408 0.507
236 This is consistent with the magnitudes of the molecular electrostatic potentials (MEPs) calculated for guanines in these three types of nucleic acid [38], and is consistent with the view that the major contributor to the observed values of k~k given in Table II and kz in Table VI will be alkylation of N7 of guanine. It should be remembered that the values of k~,k are composite rate constants for alkylation by both mustards and monomustards, while the k: values are composites of both hydrolysis and alkylation. Table VI also shows that the extent of hydrolysis of the mustards in the presence of double-stranded DNA is dependent upon the DNA basepalr ratio, with the extent of hydrolysis decreasing with the percentage of AT basepairs in the nucleic acid (Fig. 3). Since the AT regions are the preferred binding site for minor groove binders such as 3--6, the results suggest that such binding provides some protection from hydrolysis. In our previous studies [18] we formulated a scheme similar to that shown in Scheme 1 to account for the hydrolytic and alkylating reactivity of unprotonated nitrogen mustards. This scheme leads to the rate Eqn. 13 in which [Nu] represents the concentration of nucleic acid. Following on the discussion below we show the mustard binding to guanine as the nucleophile. ks
kl(k2 + k3[Nu]) k_l[Cl-] + k2 + ka[Nu]
=
(13)
and when k_l[C1] ,~ k2 + k3[Nu], ks = kl. This mechanism is confirmed by the present results, since the value of kz obtained in the presence of excess nucleic acid but a constant concentration of sodium ions is almost independent of the nature of the nucleic acid, poly(dGdC) or poly(dAdT).
50 poly dGdC
40 ZR (n ul
o "0 >.,
3O 20
-r-
10 0 0
20
40
60
80
100 poly dAdT
AT (%) Fig. 3. Plot showing a linear correlation between the percent hydrolysis of mustard 3 in the presence of a variety of nucleic acids (2.6 mM vase pair) against the percent AT in those acids.
237 At- N / CI'12C-~12cI \ CH2CH2CI
Cl
"~c~2a
CI"I2CH20H At- N" \ CH2CH2CI \ CH2CH2C1
Ar-N/CHacH2OI'I . _ _ _ _ ~ ~ ~ ~ . . _ \ CH2C~OH
At- N ~-\ CH2C~O~
Scheme 1.
Table VIII gives the values of kr obtained during alkylation of double-stranded calf thymus DNA in 0.01 M HEPES buffer at pH 7.0 by mustards 3--6 over the temperature range 25-60°C and Table IX gives the derived Arrhenius parameters, which are very similar to those found for alkylation of 4~4'nitrobenzyl)pyridine by these mustards in 50% aqueous acetone [18l. Although entropies of activation better characterize the activation process than do rates and activation energies, they still do not provide an unambiguous guide to mechanism. The observed rate constant, kv is a composite quantity, as are the activation parameters. Moreover, the rate constants have not been corrected for partial protonation of the mustards in the buffer medium. Since the temperature
238 TABLE VIII RATE CONSTANTS (kr_)FOR LOSS OF MUSTARDS BY HYDROLYSIS AND/OR ALKYLATION OF DOUBLE-STRANDED CALF THYMUS DNA IN 0.01 M HEPES, pH 7.0 Mustard
104k~ (s- 1)
3 4 5 6
25°C
37°C
50°C
60°C
0.479 0.332 0.156
2.88 1.54 0.639 0.0140
10.5 7.85 3.32 0.0428
23.9 15.2 12.7 0.282
dependence of pK~ is not known, a dissection of AH* and AS* into equilibrium and kinetic contributions is not possible, and the mechanistic assignments based on these parameters must remain tentative at best. Nevertheless, the negative values of AS* give support to a hypothesis that the first (rate determining) step in the hydrolysis pathway, k~ in Scheme 1, is a typical (internal) SN2 reaction.
Relationships between DNA binding and DNA alkylation Comparison of the values of k~ (Table VI) for the sum of the rate constants of hydrolysis and alkylation and of K (Table III) for binding of 3 - - 6 to calf thymus DNA confirms that reactivity decreases as binding becomes tighter. The values of pK. decrease in the order O, CONH > CH 2 > CO [18], which is the reverse of the binding order. A change in solvent to 0.01 M H E P E S buffer causes a slight increase in the value of k~ for 3 (2.88 x 10 -4 s -1 at 37°C (Table VII) compared with 2.62 x 10 -4 s -1 in water (Table VI)) but a large decrease in the binding constant K (0.635 x 10 e M -1 in buffer, 3.36 × 10 e M -1 in H 2 0 (Table III)). However, the rate constants of hydrolysis alone for 3 also decrease with a change in buffer medium (k1 ffi 2.25 x 10 -4 s -1, k 2 ffi 2.26 x 10 -4 s -1 in 0.01 M H E P E S compared with kl ffi 2.71 x 10 -4 s -1, k z ffi 3.40 x 10 -4 s -1 in H 2 0 ) and the small increase observed in the composite rate constant k~ reflects the diminished contribution from hydrolysis. Similar trends are observed for mustards 4 - - 6 . TABLE IX ARRHENIUS PARAMETERS DERIVED FROM k~ VALUES FOR LOSS OF MUSTARDS 3 TO 6 BY HYDROLYSIS AND/OR ALKYLATION OF DOUBLE-STRANDED CALF THYMUS DNA IN 0.01 M HEPES, pH 7.0 Mustard
Ea(kJmol-1 )
lnA
hHt(kJmo1-1)
~ ( J m o 1 - 1 K -1)
3 4 5
92 92 103 110
17 27 30 29
89 90 101 107
-26 -29 -0.14
6
-12
239 TABLE X V A L U E S O F T H E INTRINSIC ASSOCIATION C O N S T A N T (K) A N D T H E SITE SIZE 9 (n')F O R BINDING O F T H E DIOLS, F O R M E D F R O M H Y D R O L Y S I S OF M U S T A R D S 3 TO 6, TO DOUBLES T R A N D E D C A L F T H Y M U S D N A A T 25°C Diol from hydrolysis of
lO-eK
3 4 5 6
1.00 1.13 1.10 1.17
(M-1)
(0.93) a (1.07) a (1.00) ~ (1.18) a
~,'
11.1 (11.5) a 10.5 (10.6) a 10.3 (9.7) a 10.2 (10.0) a
aValues in parentheses determined by McGhee and yon Hippel model; other values determined by Scatchard model.
The final hydrolysis product of a nitrogen mustard is the diol. Comparison of the values for the binding constants of the diols of compounds 3--6 to calf thymus DNA given in Table X with those for the parent mustards (Table III) shows that the diols bind 3--4-fold less strongly. The reason for this is not entirely clear. Since --OH is electron-releasing while --Cl is electron attracting, the diols might be expected to be slightly stronger bases, which would be expected to increase binding. There is close agreement in the values of K and n ' determined using both the Scatchard and McGhee and yon Hippel models. CONCLUSIONS
The sequence-dependence of the binding constants and the thermodynamic binding parameters suggest that the quinolinium mustards 3--6 do bind to DNA by lodgement in the minor groove, preferentially in AT-rich regions. This binding serves to protect the mustard from hydrolysis. However, the primary site of alkylation appears to be on guanine. Rates of alkylation decrease as the binding constant increases, suggesting that the binding and alkylation sites may be different. While it is likely that the primary alkylation site is guanine N7, that cannot be decided by the present data. ACKNOWLEDGEMENTS
W e thank T.J. Boritzkifor helpfuladvice and A. Kucernak for developing the RF540 spectrofluorometrickineticsoftware. W e acknowledge support from the Research Committees of the N e w Zealand Universities'Grants Committee and the University of Auckland. REFERENCES 1 M. Ochoa, Alkylating agents in clinicalcancer chemotherapy, Ann. N Y Acad. Sci.,163 (1969) 921--930. 2 S.T. Garcia, A. McQuillan and L. Panasci, Correlation between the cytotoxicityof melphalan
240
3 4 5 6 7
8
9
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11
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14 15 16
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20 21
22 23 24
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25 26 27 28 29 30 31 ~i ~ •
~
1 33 34
35 36 37 38
with nucleic acids: influence of ionic strength and DNA base composition, Nucleic Acid Res., 17 (1989) 9933--9946. A.M. Jeffrey, DNA modification by chemical carcinogens, Pharmacol. Ther., 28 (1985) 237--272. K. Hemminki, Fluorescence properties of alkylated guanine derivatives, Acta Chem. Scand., B34 (1980) 603--605. M.T. Record Jr., T.M. Lohman and P. Dellaseth, Ion effects on ligand-nucleic acid interactions, J. Mol. Biol., 107 (1976) 145--158. M.T. Record Jr., C.F. Anderson and T.M. Lohman, Thermodynamic analysis of ion effects on the binding and conformational equilibria of proteins and nucleic acids: the roles of ion associatibn or release, screening, and ion effects on water activity, Q. Rev. Biophys., 11 (1978) 103--178. W.P. Wilson and J.G. Lopp, Analysis of cooperativityand ion effects in the interaction of quinacrine with DNA, Biopolymers, 18 (1979) 3025--3041. W.C.J. Ross, Arylhalogenoalkylamines, J. Chem. Soc., (1949) 183--191. A. Panthananickal, C. Hansch, A. Leo and F.R. Quinn, Structure-activity relationships in antitumor aniline mustards, J. Med. Chem., 21 (1978) 16--26. F. Quadrifoglio, V. Crescenzi and V. Giancotti, On the binding of proflavine to DNA, Biophys. C h e m . , 1 (1974) 319--324. F. Quadrifoglio and V. Crescenzi, On the binding of actinomycin and of daunomycin to DNA: ' A calorimetric and spectroscopic investigation, Biophys. Chem., 2 (1974) 64--69. K.J. Breslauer, R. Ferrante, L.A. Marky, P.B. Dervan and R.S. Youngquist, The origins of DNA binding affinity and specificity of minor groove directed ligands: correlations of thermodynamic and structural data, in: R.H. Sharma and M.H. Sharma (Eds.), Structure and Expression, Vol. 2: DNA and its Drug Complexes, Adenine Press, NY, 1985, pp. 273--290. J.B. Le Pecq and C. Paoletti, A fluorescent complex between ethidium bromide and nucleic acids, J. M o l . Biol., 27 (1967) 87--106. W. Muller and D.M. Crothers, Binding of actinomycin and related compounds to DNA, J. Mol. Biol., 35 (1968) 251--290. B. Singer, Reaction of guanosine with alkylating agents, Biochemistry, 11 (1972) 3939--3947. A. Pullman and B. Pullman, Molecular electrostatic potential of the nucleic acids, Q. Rev. Biophys., 14 (1981) 289--380.
Chem.-Biol. Interactions, 77 (1991) 223--241 Elsevier Scientific Publishers Ireland Ltd.
ALKYLATION OF NUCLEIC ACIDS BY DNA-TARGETED 4-ANILINOQUINOLINIUM ANILINE MUSTARDS : KINETIC STUDIES
CHARMIAN
J. O ' C O N N O R a, W I L L I A M A. D E N N Y b and JUN-YAO F A N a
aDepa~tment of Chemistry and bcct~bCerResearch L ~ , Auckland, P~i~;te Bog, Auckland (New Zealand)
School of Medicine, U~iversity of
(Received July 31st, 1990) (Revision received October 19th, 1990) (Accepted October 19th, 1990)
SUMMARY
The rate constants for hydrolysis of a series of 4-substituted aniline mustards Ar-X~pC~H4-N(CH2CH 2C1)2, where Ar is 4-anilinoquinolinium and X = O, CH2, CONH and CO, have been measured in water and 0.02 M irnidazole buffer at 37°C and in 50% aqueous acetone at 66°C. The equilibrium binding constants of the compounds and their hydrolysis products to nucleic acids of differing base composition have been determined at varying ionic strengths, and the results are consistent with the compounds binding as expected in the DNA minor groove. The alkylating reactivity of the mustards towards these nucleic acids has been measured in water at 37°C and in 0.01 M HEPES buffer over a range of temperatures from 25°C to 60°C. Evaluation of the thermodynamic parameters for these kinetic and equilibrium studies suggests that the interaction with nucleic acids is via an internal SN2 mechanism involving an aziridinium ion.
Key wo~ds: Aromatic nitrogen mustards -- Nucleic acids -- Entropy of activation -- Alkylation -- Association constants -- Binding
INTRODUCTION
Bifunctional alkylating agents are an important class of anticancer drugs [1], which express their cytotoxic and antitumor effectsby cross-linkingcellularDNA [2].The most common alkylatingmoiety employed isthe N-bis(2-chloroethyl)aniline functionality. Although the cytotoxic (and therapeutic) effects of these compounds are due to the formation of DNA cross-links (primarily between guanine N7 posiCorrespondence to:Charmian J. O'Connor, Department of Chemistry, Universityof Auckland, Private Bag, Auckland, N e w Zealand.
000~2797/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd.
224 tions [3]), the degree of cross-linking by simple mustards is low. The majority of DNA alkylation lesions are monoadducts, which are considered to be genotoxic (mutagenic) rather than cytotoxic [4]. There is interest in the concept [5,6] of targeting alkylating agents to DNA by attaching them to DNA-affinic carrier ligands, either intercalators [7--9] or minor groove binding moieties [10,11]. The aims of such an approach include increasing drug potency [7,12], avoiding some of the common mechanisms of cellular resistance to alkylating agents [13], and altering the pattern of DNA lesions formed [14,15] and their repair. Recent results demonstrate the usefulness of this approach, which has been shown to increase drastically the in vitro cytotoxicity of simple aniline mustards [7,11] and also significantly alter the regioselectivity of their alkylation of DNA [11,16]. In particular, targeting with minor groove binding ligands has been shown to direct away from the guanine N7 site preferred by simple alkylators to the adenine N3 site in the minor groove [11]. We have recently reported the synthesis and biological evaluation [17], and the kinetics of hydrolysis and alkylation of 4-(4'-nitrobenzyl)pyridine [18], of a series of aniline mustards (1) based on the DNA minor groove binding [19] 4-anilinoquinoline derivative (2). However, these compounds were not sufficiently water-soluble for DNA studies. In order to ascertain in more detail the mode of interaction of compounds of this general class with DNA, we prepared the more soluble quinolinium quaternary salts (3--6), and now report studies of the interaction of these compounds with nucleic acids. We have studied the kinetics of their alkylation of a variety of native nucleic acids, and have measured their equilibrium binding constants over a range of temperatures and ionic strengths. A minimum of two parameters, the intrinsic association constant, K, and the density of binding sites in the polynucleotide, n ' , is required to describe the interaction. The values of these parameters are sometimes dependent upon the model chosen to describe the interaction. In this investigation we have used both the Scatchard [20] and the McGhee and von Hippel [21] models and find the results therefrom very comparable. MATERIALS AND METHODS
Materials Double-stranded calf thymus DNA, yeast type VI RNA and synthetic poly(dGdC) and poly(dAdT) were purchased from Sigma. Micrococcus lysodeikticus DNA was purchased from Miles Laboratory Inc. Denatured DNA was prepared immediately before use by boiling the native DNA for 5 min and then cooling rapidly on ice. The concentration of nucleic acid was determined spectrophotometrically. Molar absorptivities at 260 nm are reported as 6600 M-1 cm-1 for calf thymus and M. lysodeikticus DNA [22], 8160 M -1 cm -~ for RNA [23], and 7100 M -~ cm -~ and 6600 M -1 cm -~ for poly(dGdC) and poly(dAdT), respectively [24]. Imidazole was from BDH and N-2-hydroxyethylpiperazin-N'-2-hydroxyethylpiperazin-N'ethanesulfonic acid, HEPES, was from Serva. The pH of HEPES buffer solutions was adjusted with HC104.
225
l~I/ CH2CH2Cl
~ I
HCl
1: X=O,CH2,CON~I,CO
t
CH3 2
~
~
~/~
/ CH2CH2cl
~
CH2CH2Cl
I
M~F3SO3
x 3
O
4
S
CH2 CONH
6
co
The trifluoromethanesulfonate salts of the quinolinium mustards (3--6) were prepared as described elsewhere [17]. Stock solutions were made up in methanol, and were stored at - 15°C. The initial (bifunctional) N,N-his(2-chloroethyl) compounds are referred to below as 'mustards', the half-hydrolysed (monofunctional) N(2-chloroethyl)-N(2-hydroxyethyl) compounds as 'monomustards' and the N ~ rbis(2-hydroxyethyl) compounds as 'diols'.
Apparatus Absorption spectra were measured on a Cary 219 spectrophotometer and fluorescence spectra on a Shimadzu RF540 spectrofluorophotometer, both interfaced to an Apple IIe computer. ~ P L C measurements were made using a Micromeritics 7600 HPLC Gradient System with a C-18 10.0 ~m reverse phase 250 x 5.0 mm column, and were analyzed using a Digital Solutions Delta Junior chromatography data system.
226
Rate constants of hydrolysis of mustards A 100-~1 sample of 0.16 mM mustard in methanol was added to 1 ml distilled water held at 37°C. At various time intervals aliquots (20-~1) were withdrawn and analyzed by HPLC at k 260 nm. The eluting solvent was a 1:4 mixture of solvent A (10% CH3CN + 90% buffer (1 M NH4OAc + 10 mM heptane sulfonate + 10 mM triethylamine, pH 5.00)) and solvent B (90% MeOH + 10% CH3CN) and the flow rate was 0.5 ml min -1. The rate constants kl and k2, for hydrolysis of the mustard to monomustard and of monomustard to diol, respectively, were calculated from computation of the peak areas of the three species. Details of the analysis are presented elsewhere [18]. The hydrolytic reaction was also carried out in 0.02 M imidazole buffer at ionic strength 0.025. The pH was adjusted with HC104 and the ionic strength maintained with NaC104. Under these conditions the hydrolytic products tend to precipitate (as seen by diminished HPLC peaks and changed absorbance spectra) and thus only the value of k I could be calculated. The retention times of the mustards and their hydrolysis products are given in Table I. These show unequivocally that, in completely degraded solutions of the mustards, both in water alone and in buffer systems, there is no peak corresponding to the retention time of the mustards. In other words, there is no interference from degradation products with the assay for intact mustards.
Alkylation of nucleic acids by mustards A 100-~1 sample of 0.16 mM nitrogen mustard in methanol was added to 1 ml of an aqueous solution (milli-Q water) of nucleic acid whose concentration had been determined from its absorbance at 260 nm [22,23]. The reaction was carried out at 37°C. During the incubation period (calculated to allow for 100% hydrolysis in the absence of nucleic acid) 100-~l aliquots were withdrawn, mixed well with an equal volume of butanol and centrifuged for a few seconds to separate the two phases. A 20 ~l sample of the upper {butanol) phase was then analyzed by HPLC using the same 1:4 mixture of solvent A and solvent B described above. This separation procedure removed nucleic acid components from the injection phase. During the course of the reaction three peaks (for mustard, half-mustard and diol) were observed, but the total peak area decreased rapidly. Division of the peak area of the diol at the end of the reaction by the initial peak area of the TABLE I RETENTION TIMES OF MUSTARDS AND THEIR HYDROLYSIS PRODUCTS a Compound
Mustard (min)
Monomustard (min)
Diol (min)
3 4 5 6
17.0 12.6 13.7 15.6
12.6 10.3 11.4 12.9
11.2 9.5 9.6 10.2
aRetention time of solvent 0auffer/water/MeOH) = 7.5--8.3 min.
227 mustard gave a value for extent of hydrolysis. This calculation depends on the assumptions that both the extraction coefficients and the UV absorbances of mustard and diol are equal. Calculation of the rate constant for loss of mustard, k~, gives the sum of the rate constants for hydrolysis and alkylation of nucleic acid. Rate constants of formation of alkylated products Alkylation of certain DNA bases by nitrogen mustards produces fluorescent products [25,26], and a binding assay was devised which was based on the change in fluorescence due to the DNA adducts. Suitable emission and excitation wavelengths were chosen by comparison of the spectrum of unreacted sample with that of a sample where the mustard had been allowed to react completely with the nucleic acid (5 h at 37°C, 300 ~l of 0.16 mM mustard in MeOH added to 3 ml nucleic acid). The rate of formation of aikylated products was then monitored throughout the reaction period and the experimental data treated by RF-540 spectrofluorophotometer control software to calculate values of k~k. Spectrophotometric titrations To 3 ml of a 1-~M solution of drug (mustard or diol, the latter being formed by complete hydrolysis of the corresponding mustard in milli-Q water and monitoring by HPLC) in a cuvette were added, sequentially, - 2-~1 aliquots of a concentrated solution ( - 1.2 mM basepair) of nucleic acid containing the drug at the same concentration as that in the cuvette. The total drug concentration was thus maintained at a constant value. After addition of each aliquot the absorption spectrum (220--500 nm) was recorded on a Cary 219 spectrophotometer. Addition of the aliquots was continued until either the absorbance became constant or the increasing concentration of nucleic acid itself caused a slight increase in absorbance. These titrations were repeated over a range of temperatures for binding of mustards 3-6 and at 25°C for binding of the corresponding diols of 3-6 to doublestranded calf thymus DNA, and at 19°C for binding of 3 to a number of other nucleic acids. The maximum amount of hydrolysis that would have occurred during these titrations was 5%. The Scatchard model The Scatchard [20] equation: v/C~ = Kv + Kin'
(1)
in which v is the binding ratio, C~ the concentration of free ligand, K the intrinsic association constant for the binding of the ligand to a single site in the lattice and n' the site size expressed in nucleotide residues in terms of base pairs, v and C~, determined from spectrophotometric titrations, were calculated by use of Eqns. 2:--4. (75 ffi (Af - Ax)Ct/(Af - Ab)
(2)
228 v
= Cb/N
(3)
Cb
Cf-- C t -
(4)
where Cb and Ct are the concentrations of drug bound to nucleic acid and total drug, respectively; N is the concentration of nucleic acid; and At, A b and A Xare the absorbances of the free and bound drug and the test sample, respectively. Scatchard plots of v/Cf against v yield a gradient equal to K and an x-intercept equal to 1In' The McGhee and yon Hippel model This model is used in situations where a bound ligand covers two or more lattice residues. The simple Scatchard relationship [21] does not allow for this complication since it is valid if the ligand binds to only one repeating unit, e.g. to one base or one base-pair. The McGhee and von Hippel analysis (Eqn. 5) ~ / c f --
K(1 - n'~) n' [1 -
(n'
-
1)p] n ' - I
(5)
allows for the effect of neighbouring site exclusion on binding equilibria. When n' > 1, a plot of p/C~ against v will be curved and the values of K and 1/n' are determined from the intercepts of the curve on the y- and x-axes, respectively. Calculation of effective electric charge on mustard 3 The method of Record et al. [27,28] (Eqn. 6) uses 6InK 61n[Na+]
-Z¢
(6)
in whichK is the observedequilibriumconstant for bindingof 3 to double-stranded calf thymus DNA, [Na+] is the sodiumion concentration of NaCI or NaCIO, and ¢ is the fraction of counter ions associated with each DNA phosphate. A plot of InK against ln[Na+] has a slope of -Z¢ where Z is the effective electric charge on the drug. For doublestranded DNA, ¢ -- 0.88.The alternativemethodof Wilson and Lopp [29] (Eqn.7) 5InK -- - 2 n ' ( ¢ - ¢*) - Z*¢ 51n[Na ÷]
(7)
was also used to calculate Z*, the effective electric charge on 3. In this model, n" is the number of neighbouring sites excluded upon ligand binding and ¢* is the fraction of counter ion per phosphate in the intercalated drug-DNA complex. Average values of n' = 3.0 antiC* -- 0.82 have been used in this investigation. RESULTS AND DISCUSSION
Rate constants of hydrolysis in the absence of nucleic acid While there have been extensive studies of the hydrolysis rates of aniline
229
mustards [7,30], including the non
(8)
logk2 = -3.90% - 4.30 n = 4, r = -0.92
(9)
The substantial negative slopes of these lines are indicative of assistance by electron-donation to the reaction centre. The slopes of equations describing hydrolysis of the hydrochloride salts at 66 °C in 50% aqueous acetone were comparable (-2.56 and -2.78 for k 1 and k 2 respectively). The rate constants for the first hydrolysis step (mustard to monomustard) were independent of pH in the range 6.35--7.25 in imidazole buffer, where the pH was adjusted by addition of HCIO 4 while maintaining constant ionic strength with added NaCIO 4. Measurement of the rate constant for the second hydrolysis step (monomustard to diol) was not possible because of precipitation of the hydrolysis products in the buffer medium as the reaction proceeded.
Binding to nucleic acids Association constants for binding to nucleic acids were measured specT A B L E II R A T E CONSTANTS OF HYDROLYSIS OF M U S T A R D S 3 TO 6 AT 37°C (AND OF THEIR C O R R E S P O N D I N G H Y D R O C H L O R I D E SALTS IN 50% A Q U E O U S A C E T O N E AT 66°C |18]) Compound
Solvent
pH
104 k I (s-1)
3 3 3 3 3 4 5 6
buffera buffera buffera buffera H20 H20 H20 H20
7.25 6.95 6.65 6.35
1.91 (3.69) 1.93 (3.59) 1.84 (3.68) 1.92 (3.62) 2.71 (3.15) 2.03 (2.79) 1.48 (3.26) 0.0146 (0.0353)
80.02 M imidazolebuffer, ion strength (NaClO4) ffi 0.025.
104 k 2 (s-i)
3.40 (2.50) 1.49 (2.79) 1.94 (7.01) 0.00365 (0.0694)
230
trophotometrically. Figure la shows a set of typical spectra obtained for the titration of 3 with calf thymus DNA. Addition of nucleic acid causes a bathochromic shift and a decrease in absorbance at km~ (350 nm) of the drug (Fig. lb). The presence of a clear isosbestic point in Fig. la provides further evidence for the formation of a drug/nucleic acid complex. The binding ratio ~ (tool drug bound/mol base pairs), and the free drug concentration C~were determined for mustards 3--6 in the presence of varying concentrations of calf thymus DNA at temperatures 19°C, 25°C and 37°C and in HEPES buffer at 22 °C and for mustard 3 in the presence of a variety of nucleic acids at 19°C. The results for typical binding isotherms for 3 are shown in Fig. 2. The
a ta. m9 -d • . ee ,.] ~_;a. BT"J
l,k?
I'¢//2"%
l,¢/// u
1
~.RI!
4eQ Ni.vml Inl~lth(nm) 0.800 b
i
0.60C
Ih w
0.400
0
I
10
I
20
i v
A v
,
30
A w
,
40
T
5O
Fig. 1. (a) Spectra of 3 (1 ~M) obtained upon titration with 2-~1 aliquots of 2 mM calf thymus DNA. (b) Plot showing the effect of increasing concentration of calf thymus DNA on the absorbance of 3 at X 350 nm.
231 3
d 2
1
I
I
b ~
2
0
I
I
!
.
I
I
,
u
!
I
C
f
1
a
0.1
0.2
0.3
V
0.4
0
0.1
O
g
0.2
0.3
"
I
0.4
,
0.5
V
Fig. 2. Scatehard plots for binding of 3 to nucleic acids: (a) double-stranded calf thymus DNA; (b) single-stranded calf thymus DNA; (c) double-strandedM. lysode~lct~s DNA; (d) yeast RNA; (e) po-
iy(dADT) and (f) poly(dGdC).
solidlinesdrawn through the data represent the best fitusing the Scatchard equation (Eqn. 1) [20].Parameters used to fitthis equation to the experimental data are provided in Table III. The interpretation of such data depends on the theoretical model used. Since the simplest situation [20] is considered in these studies,and the data are assumed to pertain to simple mass-action interactionbetween the drug and discretenoninteracting sites spaced along the nucleic acid chain, straight lines can be fitted (e.g. by the method of least squares) to the data points and the interaction parameters K and n' can be calculated.Under identicalbinding conditions,3 binds more strongly to double-stranded calf thymus D N A than it does to the singlestranded nucleicacid.There is also a base sequence preference, since 3 binds most strongly to poly(dAdT), least strongly to poly(dGdC), and more tightly to calf thymus DNA (50°70AT) than to M. lysodeikticus DNA (3007oAT). This is evidence that the mustards 3--6 are indeed minor groove binding ligands like the parent molecule (2), since such compounds bind preferentially to AT-rich regions in doublestranded DNA [19]. The drug binding site size n' remains essentially constant
3
3
Single-stranded calf thymus DNA
Double-stranded
.2.50 4.23 2.39
3.11
1.68
3.36 4.54 3.46 5.18
(M- 1)
19°C 10-SK
(4.0)c 2.4 2.2
2.7
(2.8) c
3.0 3.3 2.9 3.0
n ' (n)
aValues determined using the McGhee and yon Hippel model. bValues determined in 0.01 M Hepes buffer at pH 7.0. CThe site size expressed in terms of base nucleotide residues.
Yeast RNA poly(dAdT) poly(dGdC)
DNA
3 3 3
3 4 5 6
Double-stranded calf thymus DNA
M. lysodeikticus
Drug
Nucleic acid
3.01 5.88 4.32
4.89
2.21
4.99 5.55 5.09 6.72
(M 1)
19°C a 10-6//"
(3.8)¢ 2.2 2.1
2.4
(3.1) ¢
3.3 3.4 3.2 3.3
n' 2.86 3.83 3.00 4.11
(M 1)
25°C 10-SK 2.9 2.8 2.9 2.8
n' 2.17 3.00 2.46 3.14
(iV[- 1)
37°C 10-SK
2.4 2.4 2.8 3.3
n'
0.635 1.03 0.694 1.11
(M- 1)
22°C b 10-SK
The parameters were determined at all temperatures using the Scatchard model and at 19°C using the McGhee and yon Hippel model.
2.2 2.6 2.7 2.9
VALUES OF THE INTRINSIC ASSOCIATION CONSTANT (K) AND THE SITE SIZE E X P R E S S E D IN NUCLEOTIDE RESIDUES ( n ' ) FOR BINDING OF MUSTARDS TO NUCLEIC ACIDS
TABLE III
O~ t~
233 TABLE IV THERMODYNAMIC PARAMETERS DERIVED FROM E F F E C T OF TEMPERATURE ON THE INTRINSIC ASSOCIATION CONSTANT FOR BINDING OF MUSTARDS TO DOUBLESTRANDED CALF THYMUS DNA Drug
AH ° (kJ tool -1)
AG ° (kJ mo1-1)
AS ° (J mo1-1 K -1)
3 4 5 6
-18 -17 -14 -20
-37 -38 -37 -38
62 68 77 58
at 2.6 ± 0.4 nucleotides per binding site with all the nucleic acids, suggesting no change in the mode of binding. Table III also gives values of the binding parameters of these drugs to nucleic acids at 19°C determined using the McGhee and yon Hippel equation (Eqn. 5). These values of K (although a little larger) show similar trends to those calculated from the Scatchard model and the values of n are very comparable (2.7 ± 0.6). For the compounds binding to calf thymus DNA plots of lnK (K determined using the Scatchard model) against 1/T were linear, and from these van't Hoff plots values of the enthalpy of interaction, zk/-/°, were calculated (Table IV). The values of ~-/° and the derived values of the Gibbs free energy of activation, ~G ° were negative for all the mustards 3-6. Other work has shown that both DNA intercalating agents (e.g. ethidium bromide, proflavin [32] and daunomycin [33]) and minor groove binding agents (e.g. netropsin and analogues [34l) show negative enthalpies of binding. The values for the entropy of binding of 3-6 with DNA are very large and positive (Table IV). This is in agreement with other studies of minor groove binders [34], and is consistent with the large binding constants. It is well-known that raising the ionic strength of the medium decreases the value of the binding constant of small molecules to DNA [22--24,35,36]. The values of K given in Table V show that this decrease is also true for 3 when bound to TABLE V E F F E C T OF IONIC STRENGTH ON THE INTRINSIC ASSOCIATION CONSTANT FOR BINDING OF 3 TO DOUBLE-STRANDED CALF THYMUS DNA AT 20°C [ Salt] (M)
10- 5K (M - 1)
Z
Z*
0.025 M NaC1 0.010 M NaCl 0.005 M NaCl
2.68 7.64 12.0
1.1
0.66
0.025 M NaCIO 4 0.010 M NaCIO 4 0.005 M NaCIO 4
0.641 2.48 13.6
2.1
1.7
234
calf thymus DNA. Analyzing these data by the method of Record et al. [27,28] or the method of Wilson and Lopp [29] allows calculation of the value of the effective electric charge on the drug, Z or Z*, respectively, and these are given in Table V. The two models provide consistent results, demonstrating that the effective charge on the drug when it binds to the nucleic acid is dependent upon the salt which is used to adjust the ionic strength. In the presence of NaC1, the drug carries a charge between 0.66 and 1.1, but when NaClo 4 is used the charge is between 1.7 and 2.1. The compounds are methylquinolinium salts, and thus carry one permanent charge on the quinoline nitrogen. We have previously shown [18] that chloride ions markedly inhibit the formation of the aziridinium ion, thereby preventing an increase in effective electric charge. Conversely, in the presence of perchlorate ions, the values of Z are close to 2, indicating that under these conditions formation of a partial positive charge on the aziridinium ion is promoted.
Rate constants for alkylation of nucleic acids Table VI shows the derived rate constants kr for loss of the mustards in the presence of a variety of nucleic acids, measured by HPLC. The values of k~ are equal to the sum of the rate constants for alkylation of the nucleic acid and hydrolysis of the mustard, since they are derived from the loss of area in the mustard HPLC peak as the reaction progressed. Also given are the values for the percentage hydrolysis occurring at the completion of the reaction. As with TABLE VI RATE CONSTANTS FOR LOSS OF MUSTARD BY HYDROLYSIS AND/OR ALKYLATION (kr) AND VALUES FOR THE EXTENT OF HYDROLYSIS OF MUSTARDS IN NUCLEIC ACID SOLUTIONS AT 37°C Nucleic acid
Double-stranded calf thymus DNA a Single-stranded calf thymus DNA a Double-stranded M. lysodeikticus DNA b Single-stranded M. lysode/kt/cus DNA b Yeast RNA c poly(dAdT)d poly(dGdC)d a[DNA] b[DNA] C[RNA} d[dAdT]
104k~ (s-1) hydrolysis (%) 104k~ (s-1) hydrolysis (%) 104kE (s- 1) hydrolysis (%) 104kr (s-1) hydrolysis (%) 104kE (s-1) hydrolysis (%) 104k~ (s- 1) hydrolysis (%) 104kr (s- 1) hydrolysis (%)
= 0.70 mM base. ffi 0.58 mM base. ffi 1.0 mM base. ffi [dGdC] = 0.52 mM base.
3
4
5
2.62 12.4 0.869 10.9 1.85 25.0 1.94 14.3 1.80 4.2 2.34 0 2.44 39.0
0.565 16.8 0.228 8.8 1.23 31.9 1.09 15.1 1.00 1.4
0.887 15.2 0.690 9.7 1.46 26.7 1.24 13.2 0.775 2.0
6
0.0106 9.9
0.0329 13.8
0.509 0.2
235 the values of the rate constants for hydrolysis alone (k1 and k2), the values of k r also correlate well with Hammett parameters, giving in fact almost identical equations (Eqns. 10 and 11). For hydrolysis/alkylation of double-stranded calf thymus DNA (AT/GC ratio 50:50) in water:
(10)
logkr ffi -2.94% - 4.42 n = 4, r ffi - 0 . 9 2
while for double-stranded M. lysodeikticus DNA (AT/GC ratio 30:70) in water:
(11)
logkr = - 2 . 4 1 % - 4.18 n ffi 4, r = - 0 . 9 3 and for yeast RNA in water: logk, = - 0 . 3 9 % - 4.09 n ffi 4, r = - 0 . 9 7
(12)
Since alkylated adducts of guanosine [26,37] (and to a lesser extent adenosine [25]) have fluorescent properties, the formation of these adducts may be monitored by spectrofluorophotometry, as an alternative measure of DNA alkylation alone. Although this method of analysis probably does not detect reaction at other bases, it does provide information about relative rates of reaction within the series. The values obtained by this method (Table VII) for the rates of alkylation by compounds 3 and 5 agree well with those determined above by HPLC. These data show that double-stranded calf thymus DNA is alkylated about 2--3-fold as rapidly as when single-stranded, but at about the same rate as RNA.
T A B L E VII R A T E C O N S T A N T S F O R F O R M A T I O N (kALK) OF F L U O R E S C E N T DNA-ADDUCTS AT 37QC Nucleic acid
3
5
Double-stranded calf thymus DNA
)~ex (nm) )~em(nm) 104kalk (s-1)
309 410 2.09
310 405 0.654
Single-strandedcalfthymus D N A
)~ex (nm) )~em(nm) 104kalk (s- 1)
310 410 1.19
310 420 0.201
Yeast RNA
kex (nm) kern (rim) 104kalk (s- 1)
308 412 2.23
308 408 0.507
236 This is consistent with the magnitudes of the molecular electrostatic potentials (MEPs) calculated for guanines in these three types of nucleic acid [38], and is consistent with the view that the major contributor to the observed values of k~k given in Table II and kz in Table VI will be alkylation of N7 of guanine. It should be remembered that the values of k~,k are composite rate constants for alkylation by both mustards and monomustards, while the k: values are composites of both hydrolysis and alkylation. Table VI also shows that the extent of hydrolysis of the mustards in the presence of double-stranded DNA is dependent upon the DNA basepalr ratio, with the extent of hydrolysis decreasing with the percentage of AT basepairs in the nucleic acid (Fig. 3). Since the AT regions are the preferred binding site for minor groove binders such as 3--6, the results suggest that such binding provides some protection from hydrolysis. In our previous studies [18] we formulated a scheme similar to that shown in Scheme 1 to account for the hydrolytic and alkylating reactivity of unprotonated nitrogen mustards. This scheme leads to the rate Eqn. 13 in which [Nu] represents the concentration of nucleic acid. Following on the discussion below we show the mustard binding to guanine as the nucleophile. ks
kl(k2 + k3[Nu]) k_l[Cl-] + k2 + ka[Nu]
=
(13)
and when k_l[C1] ,~ k2 + k3[Nu], ks = kl. This mechanism is confirmed by the present results, since the value of kz obtained in the presence of excess nucleic acid but a constant concentration of sodium ions is almost independent of the nature of the nucleic acid, poly(dGdC) or poly(dAdT).
50 poly dGdC
40 ZR (n ul
o "0 >.,
3O 20
-r-
10 0 0
20
40
60
80
100 poly dAdT
AT (%) Fig. 3. Plot showing a linear correlation between the percent hydrolysis of mustard 3 in the presence of a variety of nucleic acids (2.6 mM vase pair) against the percent AT in those acids.
237 At- N / CI'12C-~12cI \ CH2CH2CI
Cl
"~c~2a
CI"I2CH20H At- N" \ CH2CH2CI \ CH2CH2C1
Ar-N/CHacH2OI'I . _ _ _ _ ~ ~ ~ ~ . . _ \ CH2C~OH
At- N ~-\ CH2C~O~
Scheme 1.
Table VIII gives the values of kr obtained during alkylation of double-stranded calf thymus DNA in 0.01 M HEPES buffer at pH 7.0 by mustards 3--6 over the temperature range 25-60°C and Table IX gives the derived Arrhenius parameters, which are very similar to those found for alkylation of 4~4'nitrobenzyl)pyridine by these mustards in 50% aqueous acetone [18l. Although entropies of activation better characterize the activation process than do rates and activation energies, they still do not provide an unambiguous guide to mechanism. The observed rate constant, kv is a composite quantity, as are the activation parameters. Moreover, the rate constants have not been corrected for partial protonation of the mustards in the buffer medium. Since the temperature
238 TABLE VIII RATE CONSTANTS (kr_)FOR LOSS OF MUSTARDS BY HYDROLYSIS AND/OR ALKYLATION OF DOUBLE-STRANDED CALF THYMUS DNA IN 0.01 M HEPES, pH 7.0 Mustard
104k~ (s- 1)
3 4 5 6
25°C
37°C
50°C
60°C
0.479 0.332 0.156
2.88 1.54 0.639 0.0140
10.5 7.85 3.32 0.0428
23.9 15.2 12.7 0.282
dependence of pK~ is not known, a dissection of AH* and AS* into equilibrium and kinetic contributions is not possible, and the mechanistic assignments based on these parameters must remain tentative at best. Nevertheless, the negative values of AS* give support to a hypothesis that the first (rate determining) step in the hydrolysis pathway, k~ in Scheme 1, is a typical (internal) SN2 reaction.
Relationships between DNA binding and DNA alkylation Comparison of the values of k~ (Table VI) for the sum of the rate constants of hydrolysis and alkylation and of K (Table III) for binding of 3 - - 6 to calf thymus DNA confirms that reactivity decreases as binding becomes tighter. The values of pK. decrease in the order O, CONH > CH 2 > CO [18], which is the reverse of the binding order. A change in solvent to 0.01 M H E P E S buffer causes a slight increase in the value of k~ for 3 (2.88 x 10 -4 s -1 at 37°C (Table VII) compared with 2.62 x 10 -4 s -1 in water (Table VI)) but a large decrease in the binding constant K (0.635 x 10 e M -1 in buffer, 3.36 × 10 e M -1 in H 2 0 (Table III)). However, the rate constants of hydrolysis alone for 3 also decrease with a change in buffer medium (k1 ffi 2.25 x 10 -4 s -1, k 2 ffi 2.26 x 10 -4 s -1 in 0.01 M H E P E S compared with kl ffi 2.71 x 10 -4 s -1, k z ffi 3.40 x 10 -4 s -1 in H 2 0 ) and the small increase observed in the composite rate constant k~ reflects the diminished contribution from hydrolysis. Similar trends are observed for mustards 4 - - 6 . TABLE IX ARRHENIUS PARAMETERS DERIVED FROM k~ VALUES FOR LOSS OF MUSTARDS 3 TO 6 BY HYDROLYSIS AND/OR ALKYLATION OF DOUBLE-STRANDED CALF THYMUS DNA IN 0.01 M HEPES, pH 7.0 Mustard
Ea(kJmol-1 )
lnA
hHt(kJmo1-1)
~ ( J m o 1 - 1 K -1)
3 4 5
92 92 103 110
17 27 30 29
89 90 101 107
-26 -29 -0.14
6
-12
239 TABLE X V A L U E S O F T H E INTRINSIC ASSOCIATION C O N S T A N T (K) A N D T H E SITE SIZE 9 (n')F O R BINDING O F T H E DIOLS, F O R M E D F R O M H Y D R O L Y S I S OF M U S T A R D S 3 TO 6, TO DOUBLES T R A N D E D C A L F T H Y M U S D N A A T 25°C Diol from hydrolysis of
lO-eK
3 4 5 6
1.00 1.13 1.10 1.17
(M-1)
(0.93) a (1.07) a (1.00) ~ (1.18) a
~,'
11.1 (11.5) a 10.5 (10.6) a 10.3 (9.7) a 10.2 (10.0) a
aValues in parentheses determined by McGhee and yon Hippel model; other values determined by Scatchard model.
The final hydrolysis product of a nitrogen mustard is the diol. Comparison of the values for the binding constants of the diols of compounds 3--6 to calf thymus DNA given in Table X with those for the parent mustards (Table III) shows that the diols bind 3--4-fold less strongly. The reason for this is not entirely clear. Since --OH is electron-releasing while --Cl is electron attracting, the diols might be expected to be slightly stronger bases, which would be expected to increase binding. There is close agreement in the values of K and n ' determined using both the Scatchard and McGhee and yon Hippel models. CONCLUSIONS
The sequence-dependence of the binding constants and the thermodynamic binding parameters suggest that the quinolinium mustards 3--6 do bind to DNA by lodgement in the minor groove, preferentially in AT-rich regions. This binding serves to protect the mustard from hydrolysis. However, the primary site of alkylation appears to be on guanine. Rates of alkylation decrease as the binding constant increases, suggesting that the binding and alkylation sites may be different. While it is likely that the primary alkylation site is guanine N7, that cannot be decided by the present data. ACKNOWLEDGEMENTS
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